Professional Documents
Culture Documents
WVJ13 (4), 561 570, December25,2023
WVJ13 (4), 561 570, December25,2023
WVJ13 (4), 561 570, December25,2023
DOI: https://dx.doi.org/10.54203/scil.2023.wvj60
INTRODUCTION
The success of an artificial insemination program using frozen semen depends on the quantity and quality of semen
ejaculated by a male and the freezing process. This process begins with semen collection, evaluation, dilution, filling and
sealing, equilibration, aerating over liquid nitrogen vapor, immersion in liquid nitrogen, and thawing (Bebas et al., 2018;
Bebas and Agustina, 2022).
During the semen freezing process, spermatozoa are subjected to extremely low temperatures of up to -196°C,
which affects the integrity of the cell membranes. At these low temperatures, the interior of cells undergoes physical and
chemical changes, including increased intracellular electrolyte concentrations and the formation of ice crystals, causing
cold shock (Anwar et al., 2015; Liu et al., 2021; Carriço et al., 2023). Cold shock damages the plasma and acrosome
membranes (Ax et al., 2000; Bebas et al., 2018). Another primary issue faced during semen freezing is the exposure of
spermatozoa to free radicals (Zhang et al., 2021; Bebas and Agustina, 2022). During this process, spermatozoa undergo
peroxidation that generates free radicals such as hydroxyl (-OH) and singlet oxygen or 1[O]2 (Bansal and Bilaspuri, 2011;
Park and Yu, 2017). These radicals are highly reactive and potentially induce lipid peroxidation in the plasma and
acrosome membranes (Douard et al., 2003; Bebas and Agustina, 2022). Lipid oxidation in the plasma membrane
produces malondialdehyde (MDA) as a marker for the presence of toxic free radicals, which reduces motility and causes
DNA damage (Dutta et al., 2019).
The plasma membrane regulates the influx and efflux of all electrolytes and substrates the spermatozoa needs
(Delgado-Bermúdez et al., 2022). The physiological integrity is closely associated with protecting and maintaining
spermatozoa motility during storage, within the female reproductive tract, capacitation, and fertilization. This is
particularly significant due to the direct or indirect interactions and adhesion between the plasma membrane and cumulus
oophorus (Arvioges et al., 2021; Syafi'i and Rosadi, 2022).
The acrosome membrane is a structure that covers two-thirds of the anterior head. It contains acrosin,
hyaluronidase, and corona-penetrating enzyme, which function to lyse the zona pellucida as a pathway for spermatozoa
561
To cite this paper: Bebas W, Gorda IW, Agustina KK, and Merdana IM (2023). Effects of Adding Glutathione to AndroMed Diluent on Intact Plasma and Acrosome
Membranes, and Progressive Motility of Cattle Spermatozoa During Freezing Processes. World Vet. J., 13 (4): 561-570. DOI: https://dx.doi.org/10.54203/scil.2023.wvj60
to enter the cytoplasm of the ovum during fertilization (Nofa et al., 2017). This membrane is vital in assessing
spermatozoa quality, as it is crucial for successful fertilization. Damage to the membrane leads to the leakage of enzymes
and a decrease in fertilization capacity (Cahya et al., 2017; Arvioges et al., 2021).
AndroMed is one of the commercial semen diluents devoid of egg yolk, which is prone to microbial contamination.
It is user-friendly, and the composition includes sugar, tris hydroxy-aminomethane, and glycerol as an energy source, a
buffer, and cryoprotectant, respectively, alongside antibiotics to prevent bacterial growth (Juniandri and Isnaini, 2014).
AndroMed also contains soybean extract rich in lecithin, minerals including sodium, magnesium, calcium, chloride,
phosphorus, potassium, and manganese, as well as carbohydrates, proteins, citric acid, glycerol lecithin, fats, and
glyceryl phosphoryl choline (Lestari et al., 2014; Aji et al., 2019).
Glutathione is a primary antioxidant that prevents the formation of new free radicals (Ansari et al., 2021). It
converts existing free radicals into less membrane-impactful molecules. Free radicals are atoms or molecules with one or
more unpaired electrons and are highly unstable (Zulaikhah, 2017). To obtain a pair, free radicals react with other atoms
or molecules, such as unsaturated fatty acids, proteins, nucleic acids, or lipopolysaccharides, resulting in the creation of
abnormal compounds (Pizzorno, 2014; Ansari et al., 2021). The addition of glutathione to the spermatozoa diluent
medium reduces or prevents the formation of free radicals that could damage plasma and acrosome membranes as well
as motility. Consequently, the fertility of frozen semen increases, ultimately leading to higher pregnancy rates. The
recommended concentration of glutathione suitable as a diluent for cattle semen freezing is approximately 1 mmol/L
(Syarifuddin et al., 2012), and for dairy goats is 2 mmol/L (Zou et al., 2021). Based on the information provided, this
study aimed to determine the impact of adding glutathione to AndroMed diluent on the intact plasma membrane, intact
acrosome membrane, and progressive motility of Bali cattle spermatozoa in the post-dilution, post-equilibration, and
post-thawing processes. This research reveals new information regarding the role of glutathione as an antioxidant added
to Andromed semen diluent on the quality of Bali cattle semen during the freezing process.
Ethical approval
All procedures have been reviewed by the Experimental Animal Ethics Committee, Faculty of Veterinary
Medicine, Udayana University, and have received approval No. B/252/UN14.2.9/PT.01.04/2023.
Semen evaluation
The collected semen from a single male was subjected to both macroscopic and microscopic examinations. The
research design used a completely randomized design, where 72 spermatozoa samples were divided into two treatment
groups, with three stages of the freezing process and each with six replications. The macroscopic examination entailed
evaluations of volume, pH, consistency/viscosity, color, and odor. Meanwhile, the microscopic examination using
Microscope Binoculer Olympic CX-23 (Olympus Corporation, Japan) with magnification 400x included analyses of
562
To cite this paper: Bebas W, Gorda IW, Agustina KK, and Merdana IM (2023). Effects of Adding Glutathione to AndroMed Diluent on Intact Plasma and Acrosome
Membranes, and Progressive Motility of Cattle Spermatozoa During Freezing Processes. World Vet. J., 13 (4): 561-570. DOI: https://dx.doi.org/10.54203/scil.2023.wvj60
mass motility, individual motility, spermatozoa concentration, abnormality, IPM, and IAM (Cocchia et al., 2011;
Susilawati, 2011).
Cement dilution
The collected semen and the AndroMed diluent were placed for 3-5 minutes in a water bath at a temperature of
37°C. This step was taken to equilibrate the temperature of the diluent with that of the semen. Furthermore, semen that
underwent evaluation was diluted using the diluent prepared according to the formula for calculating the diluent quantity
(Wishart, 2009; Syarifuddin et al., 2012).
Straw printing
For liquid semen that fulfilled the quality standards before freezing, the next step entailed labeling the packaging
using a straw printing machine (Minitub, Hauptstrasse-Germany) (Syarifuddin et al., 2012; Maleki et al., 2023).
Equilibration
The semen that underwent filling and sealing was then stored in a refrigerator at a temperature of 4°C for 4 hours
(Murphy et al., 2018; Swarna et al., 2023).
Freezing
The freezing process was carried out by immersing the semen product into liquid nitrogen until fully submerged,
resulting in the temperature dropping to -196°C (Ansari et al., 2021).
563
To cite this paper: Bebas W, Gorda IW, Agustina KK, and Merdana IM (2023). Effects of Adding Glutathione to AndroMed Diluent on Intact Plasma and Acrosome
Membranes, and Progressive Motility of Cattle Spermatozoa During Freezing Processes. World Vet. J., 13 (4): 561-570. DOI: https://dx.doi.org/10.54203/scil.2023.wvj60
followed by evaluation under a light microscope Binoculer Olympic CX-23 (Olympus Corporation, Japan) at 400x
magnification on a minimum of 200 spermatozoa. Spermatozoa with IPM were characterized by coiled or swelling tails,
while damaged ones exhibited straight tails.
Data analysis
The obtained data were analyzed using analysis of variance (ANOVA) through SPSS version 25 for Windows. In
cases of significant differences among treatments, further analysis was conducted using the Duncan test. P value < 0.05
is declared statistically significant.
RESULTS
The macroscopic evaluation of fresh Bali cattle semen yielded normal characteristic aroma, moderate-normal
consistency, creamy color, and pH of 6.5. Meanwhile, the microscopic evaluation showed mass motility (+++),
spermatozoa concentration of 1358x106 ml-1, 75% progressive motility, 86% viability, 5% abnormality, IPM at 86%, and
IAM at 89%. The comprehensive results of the semen evaluation are presented in Table 1. The addition of glutathione
with a concentration of 1 mmol to the AndroMed diluent led to an improvement in semen quality during the freezing
process stages, including dilution, post-equilibration, and post-thawing. Statistical analysis indicated that the treatment
and freezing stages significantly (p < 0.05) affected the quality of Bali cattle semen, compared to control (Table 2). The
effect of adding glutathione to the AndroMed diluent on the semen quality during the freezing process stages is shown in
Figures 1, 2, and 3. Microphotograph of sperm motility and integrity are shown in Figures 4, 5, and 6.
Table 1 shows that fresh Bali cattle spermatozoa have progressive motility, IAM, and IPM of 75%, 89%, and 88%
respectively. Meanwhile, Table 2 shows that the stages of the freezing process had a significant effect (p < 0.05) on
reducing progressive motility, IAM, and IPM of spermatozoa after dilution compared to post-equilibration and post-
equilibration, compared to post-thawing in all treatment groups. Next, the obtained showed that the addition of 1 mmol
gluthation to Andromed diluent had a significant difference (p < 0.05) in increasing progressive motility, IAM, and IPM
of Bali cattle spermatozoa at the post-dilution, post-equilibration, and post thawing compared to control.
Figure 1. Motility of 4 years old Bali cattle spermatozoa due to the effect of adding 1 mmol glutathione to AndroMed
diluent during the semen freezing process
564
To cite this paper: Bebas W, Gorda IW, Agustina KK, and Merdana IM (2023). Effects of Adding Glutathione to AndroMed Diluent on Intact Plasma and Acrosome
Membranes, and Progressive Motility of Cattle Spermatozoa During Freezing Processes. World Vet. J., 13 (4): 561-570. DOI: https://dx.doi.org/10.54203/scil.2023.wvj60
Figure 2. Intact acrosome membrane of 4 years old Bali cattle spermatozoa due to the effect of adding 1 mmol
glutathione to AndroMed diluent during the semen freezing process
Figure 3. Intact plasma membrane of 4 years old Bali cattle spermatozoa due to the effect of adding 1 mmol glutathione
to AndroMed diluent during the semen freezing process
Figure 4. Micropothograph of 4 years old Bali cattle Figure 5. Microphotograph of 4 years old Bali cattle
spermatozoa with normal morphology (400x). spermatozoa with intact acrosome membrane (white
arrow) and detached acrosome membrane (blue arrow,
400x)
565
To cite this paper: Bebas W, Gorda IW, Agustina KK, and Merdana IM (2023). Effects of Adding Glutathione to AndroMed Diluent on Intact Plasma and Acrosome
Membranes, and Progressive Motility of Cattle Spermatozoa During Freezing Processes. World Vet. J., 13 (4): 561-570. DOI: https://dx.doi.org/10.54203/scil.2023.wvj60
Table 1. Macroscopic and microscopic fresh semen
quality of Bali cattle aged 4 years
Parameter Value
Table 2. Effect of freezing stages on semen quality of Bali Cattle aged 4 years diluted using AndroMed with the addition
of 1 mmol Glutathione
Diluted
Parameter Freezing stages AndroMed +
AndroMed
1 mmol Glutathione
Post-Dilution 72.75 ± 1.25Aa 74.38 ± 1.15Ba
Progressive motility (%) Post-Equilibration 63.00 ± 1.82Ab 66.25 ± 0.95Bb
Past-Thawing 43.00 ± 0.81Ac 46.75 ± 0.95Bc
Post-Dilution 84.75 ± 0.95Aa 85.75 ± 1.71Ba
Ab
Intact acrosome membrane (%) Post-Equilibration 75.50 ± 1.29 77.25 ± 1.70Bb
Past-Thawing 48.50 ± 1.29Ac 55.75 ± 2.06Bc
Aa
Post-Dilution 83.75 ± 0.95 85.25 ± 0.95Ba
Intact plasma membrane (%) Post-Equilibration 76.00 ± 1.41Ab 77.25 ± 1.50Bb
Past-Thawing 54.50 ± 1.29Ac 63.75 ± 1.89Bc
abc ABC
Superscript letters that differ towards the column show a significant difference (p < 0.05); Different superscripts letters towards the row indicate
significant differences (p < 0.05)
DISCUSSION
The results from this research (Table 1) were consistent with Bebas et al. (2022), where macroscopic evaluation
such as volume, odor, consistency, color, and pH was reported as 5.98±1.35 (Bebas and Agustina, 2022). Meanwhile,
microscopic evaluation, including mass motility, concentration, progressive motility, abnormality, and IPM was noted as
+++, 1104 ± 202.21 x 106 ml-1 of ejaculate, 69.58±0.30%, 4.66±1.58%, and 83.22±1.64%, respectively. Hafez and Hafez
(2000) stated that normal semen appeared whitish-gray to pale cream in color, with a thick consistency. The consistency
correlates with the color; for instance, creamy-colored semen tends to have a thick or viscous consistency, while clear or
light-colored semen is usually less viscous (Islam et al., 2018). The distinct aroma of cattle semen indicates normalcy
and the absence of contamination. This aligned with Ilaria (2011), which categorized semen odor as distinctive.
According to Hafez and Hafez (Hafez and Hafez, 2000), the concentration of cattle spermatozoa ranges between 800-
2000 million/ml. The observed mass motility was very good (+++), characterized by large, numerous, dark, thick, active,
and fast-moving waves. In general, the cattle semen fell within the normal concentration with good quality and integrity,
in line with reports by Garner and Hafez (2000) and Prastowo et al. (2018).
During the semen freezing process, including dilution and equilibration, spermatozoa encounter extremely low and
extreme temperatures, reaching -196°C. Freezing significantly affects the cell membrane of spermatozoa, with subzero
temperatures below the freezing point causing physical and chemical changes, including the formation of ice crystals and
an increase in intracellular electrolyte concentration, resulting in cold shock (Pratiwi et al., 2014; Çelik et al., 2020).
According to previous research, cold shock culminates in damage to both plasma and acrosome membranes (Liu et al.,
2021; Carriço et al., 2023). The main challenge encountered during semen freezing was not only cold shock but also the
exposure of spermatozoa to free radical attacks such as hydroxyl and singlet oxygen (Park and Yu, 2017; Angrimani et
al., 2018). These highly reactive radicals can induce lipid peroxidation on plasma and acrosome membranes (Bansal and
566
To cite this paper: Bebas W, Gorda IW, Agustina KK, and Merdana IM (2023). Effects of Adding Glutathione to AndroMed Diluent on Intact Plasma and Acrosome
Membranes, and Progressive Motility of Cattle Spermatozoa During Freezing Processes. World Vet. J., 13 (4): 561-570. DOI: https://dx.doi.org/10.54203/scil.2023.wvj60
Bilaspuri, 2011; Syafitri et al., 2022). The spermatozoa plasma membrane consists of double lipids containing
unsaturated fatty acids which are very susceptible to peroxidation (Douard et al., 2003). Free radicals work to oxidize
lipids in the membrane so that the unsaturated fatty acid chains are broken and produce MDA, which is toxic to
spermatozoa cells (Zulaikhah, 2017; Zhang et al., 2021). The presence of MDA is an indicator of the existence of free
radicals (Dutta et al., 2019) that damage the plasma membrane, resulting in a decrease in the quality and integrity of
spermatozoa, including concentration, motility, and DNA damage (Prihantoko et al., 2022).
Research related to antioxidants as cryoprotectants has reported success in reducing oxidative damage due to
reactive oxygen species (Zulaikhah, 2017; Liu et al., 2021). Glutathione is a significant antioxidant that neutralizes free
radicals for cryopreserved in dogs (Angrimani et al., 2018), Indian red jungle fowl (Ansari et al., 2021), Mithun cattle
(Perumal et al., 2021), goat (Zou et al., 2021) and sheep (Syafitri et al., 2022). It is a naturally occurring substance
present in the body since birth and is found both within and outside cells (Zou et al., 2021). Blood glutathione levels
range from 5 to 8 mM/L, with the highest concentration being present in the liver, which serves as the most essential
organ for detoxification in the body. This antioxidant is also present in the spleen, kidneys, lungs, heart, brain, and
stomach (Zulaikhah, 2017).
The glutathione antioxidant system serves as a primary endogenous protection mechanism, actively participating in
the neutralization of reactive oxygen species (ROS) and maintaining the reduced (active) forms of vitamins C and E
(Biswas et al., 2020). Glutathione is also called the “master antioxidant” as it could lead to the activity of other
antioxidants (Chakravorty et al., 2020). For instance, vitamins C and E capture free radicals, followed by the transfer to
glutathione, which then cycles to capture more ROS (Zeitoun and Al-Damegh, 2014). This antioxidant neutralizes and
eliminates free radicals through urine. Its efficacy in protecting cells surpasses other antioxidants, including vitamins C
and E. Furthermore, glutathione protects DNA and RNA chains from degradation and shields the nucleus from free
radicals. The glutathione binds and expels unwanted substances through urine and bile (Solihati et al., 2018). The
presence of glutathione protects cells from toxic ROS effects and acts as a scavenger for hydroxyl (-OH) and superoxide
(O2-) radicals. This antioxidant also plays roles in maintaining cell survival, DNA replication, protein thiolation, enzyme
catalysis, membrane transport, receptor action, intermediary metabolism, and cell maturation (Zhang et al., 2005).
According to a previous study, glutathione captures peroxides that could harm cells. Due to the mitochondrial respiration
process, all aerobic organisms naturally face oxidative stress. Compounds, including superoxide (O 2-) and hydrogen
peroxide (H2O2), induce the production of oxygen radicals, which are toxic and capable of causing lipid peroxidation and
cellular injury (Zeitoun and Al-Damegh, 2014; Bollwein and Bittner, 2018). Based on the results, adding glutathione at a
concentration of 1 mmol to the AndroMed diluent significantly improved semen quality, including spermatozoa motility,
IPM, and IAM, during the freezing process stages.
CONCLUSION
The addition of glutathione with a concentration of 1 mmol to the AndroMed diluent significantly improved the frozen
semen quality characterized by progressive motility, intact acrosome membrane, and intact plasma membrane during the
freezing process stages, including post-dilution, post-equilibration, and post-thawing. Furthermore, research needs to be
carried out on fertility tests on semen quality through artificial insemination technology.
DECLARATIONS
Funding
This work received funding assistance from the flagship research program of the Faculty of Veterinary Medicine,
Udayana University, Bali, Indonesia with grant Number B/78.367/UN14.4.A/PT.01.03/2022.
Acknowledgments
The authors humbly express their gratitude to the Chancellor of Udayana University, the Head of the Research and
Community Service Institute, and the Dean of the Faculty of Veterinary Medicine for their support and facilities so that
the research could be completed.
Authors’ contributions
Wayan Bebas is the Conception and design of the study. Wayan Bebas and I Wayan Gorda conducted an
Acquisition of data. Wayan Bebas and I Made Merdana Analysis and interpretation of data, and wrote the manuscript.
567
To cite this paper: Bebas W, Gorda IW, Agustina KK, and Merdana IM (2023). Effects of Adding Glutathione to AndroMed Diluent on Intact Plasma and Acrosome
Membranes, and Progressive Motility of Cattle Spermatozoa During Freezing Processes. World Vet. J., 13 (4): 561-570. DOI: https://dx.doi.org/10.54203/scil.2023.wvj60
Kadek Karang Agustina and I Wayan Gorda give the Critical review and revision. All authors checked and confirmed
the data analysis and the final version of manuscript.
Competing interests
We declare there is no any conflict of interest in the publication of this article.
Ethical consideration
Ethical issues, such as data fabrication, double publication and submission, redundancy, plagiarism, consent to
publish, and misconduct, have been checked by all the authors before publication in this journal.
REFERENCES
Aji RN, Panjono, Agus A, Widyobroto BP, Hartatik T, Budisatria IGS, Ismaya, Fathoni A, Kumala S, and Bintara S (2019). The
effect of andromed® and coconut water+ 20% egg yolk as diluent on semen motility of belgian blue cattle. IOP Conference
Series: Earth and Environmental Science, 387: 012127. DOI: https://www.doi.org/10.1088/1755-1315/387/1/012127
Angrimani DSR, Nichi M, Brito MM, Kawai GKV, Rui BR, Losano JDA, Vieira NMG, Francischini MCP, Cruz DSG, Queiroz-
Hazarbassanov N et al. (2018). The use of reduced glutathione (GSH) as antioxidant for cryopreserved sperm in dogs. Arquivo
Brasileiro de Medicina Veterinária e Zootecnia, 70(2): 419-428. DOI: https://www.doi.org/10.1590/1678-4162-9684
Ansari MS, Rakha BA, Akhter S, Akhter A, Blesbois E, and Santiago-Moreno J (2021). Effect of glutathione on pre and post-freezing
sperm quality of indian red jungle fowl (Gallus gallus murghi). Theriogenology, 172: 73-79. DOI:
https://www.doi.org/10.1016/j.theriogenology.2021.06.008
Anwar P, Ondho YS, and Samsudewa D (2015). Quality of membrane plasma and acrosome integrity of bali bull preserved at 5oc in
extender sugar cane extract and addition of egg yolk. Agromedia: Berkala Ilmiah Ilmu-ilmu Pertanian, 33(1): 53-63. DOI:
https://www.doi.org/10.47728/ag.v33i1.103
Arvioges A, Anwar P, and Jiyanto J (2021). The effectiveness of thawing temperature intact plasma membrane (mpu) and intact
crosome caps (tau) of bali cattle spermatozoa. Green Swarnadwipa: Jurnal Pengembangan Ilmu Pertanian, 10(2): 342-350.
Avialable at: https://www.ejournal.uniks.ac.id/index.php/GREEN/article/view/1350
Ax RL, Dally M, Didion BA, Lenz RW, Love CC, Varner DD, Hafez B, and Bellin ME (2000). Semen evaluation. In: B. Hafez, E. S.
E. Hafez (Editors), Reproduction in farm animals, 7th Edition. Lippincont Wiliams and Wilkins, pp. 363-375. DOI:
https://www.doi.org/10.1002/9781119265306.ch25
Baharum A, Arifiantini RI, and Yusuf TL (2017). Freezing capability of pasundan bull sperm using tris-egg yolk, tris-soy, and
andromed® diluents. Jurnal Kedokteran Hewan-Indonesian, 11(1): 45-49. DOI:
https://www.doi.org/10.21157/j.ked.hewan.v11i1.5810
Bansal AK and Bilaspuri G (2011). Impacts of oxidative stress and antioxidants on semen functions. Veterinary Medicine
International, 2011: 686137. DOI: https://www.doi.org/10.4061/2011/686137
Bebas W and Agustina KK (2022). The use of lactose-astaxanthin to maintain the quality of green junglefowl frozen semen.
Biodiversitas Journal of Biological Diversity, 23(11): 5759-5764. DOI: https://www.doi.org/10.13057/biodiv/d231128
Bebas W, Gorda W, Trilaksana IGNB, Laksmi DNDI, and Pemayun TGO (2018). Lactose-astaxanthin increased the frozen semen
quality of gembrong goat in conservation efforts. Jurnal Veteriner, 19(3): 390-396. Available at:
https://ojs.unud.ac.id/index.php/jvet/article/view/31308
Biswas P, Dellanoce C, Vezzoli A, Mrakic-Sposta S, Malnati M, Beretta A, and Accinni R (2020). Antioxidant activity with increased
endogenous levels of vitamin C, E and A following dietary supplementation with a combination of glutathione and resveratrol
precursors. Nutrients, 12(11): 3224. DOI: https://www.doi.org/10.3390/nu12113224
Bollwein H and Bittner L (2018). Impacts of oxidative stress on bovine sperm function and subsequent in vitro embryo development.
Animal Reproduction, 15(Suppl 1): 703-710. DOI: https://www.doi.org/10.21451%2F1984-3143-AR2018-0041
Cahya RI, Setiatin ET, and Ondho YS (2017). Persentase membran plasma utuh dan tudung akrosom utuh spermatozoa kambing
peranakan etawah dalam pengencer yang berbeda [Percentage of intact plasma membrane and intact acrosomal cap of Etawah
crossbreed goat spermatozoa in different diluents]. Seminar Nasional Sekolah Tinggi Penyusunan Pertanian. Magelang:
Politeknik Pembangunan Pertanian Yogyakarta-Magelang, pp. 406-416. Available at:
https://jurnal.polbangtanyoma.ac.id/pros2020yoma/article/view/508
Carriço C, Barbas JP, Pimenta J, and Simões J (2023). Effect of in vitro addition of melatonin and glutathione on seminal parameters
of rams after thawing. Veterinary Science and Zoology, 10: 446. DOI: https://www.doi.org/10.20944/preprints202305.2068.v1
Çelik SC, Özekici Ü, Cincik M, Selam B, and Çakil YD (2020). Effects of antioxidants on motility and DNA integrity in frozen-
thawed sperm. Maltepe Tıp Dergisi, 12(2): 41-48. DOI: https://www.doi.org/10.35514/mtd.2020.28
Chakravorty S, Malvi A, Chaturvedi A, Sonkusare K, and Dave N (2020). Glutathione–the master antioxidant. International Journal of
Medical Research and Pharmaceutical Sciences, 7(2): 1-11. DOI: https://www.doi.org/10.5281/zenodo.3700160
Cocchia N, Pasolini M, Mancini R, Petrazzuolo O, Cristofaro I, Rosapane I, Sica A, Tortora G, Lorizio R, Paraggio G et al. (2011).
Effect of sod (Superoxide dismutase) protein supplementation in semen extenders on motility, viability, acrosome status and erk
(extracellular signal-regulated kinase) protein phosphorylation of chilled stallion spermatozoa. Theriogenology, 75(7): 1201-
1210. DOI: https://www.doi.org/10.1016/j.theriogenology.2010.11.031
568
To cite this paper: Bebas W, Gorda IW, Agustina KK, and Merdana IM (2023). Effects of Adding Glutathione to AndroMed Diluent on Intact Plasma and Acrosome
Membranes, and Progressive Motility of Cattle Spermatozoa During Freezing Processes. World Vet. J., 13 (4): 561-570. DOI: https://dx.doi.org/10.54203/scil.2023.wvj60
Delgado-Bermúdez A, Yeste M, Bonet S, and Pinart E (2022). A review on the role of bicarbonate and proton transporters during
sperm capacitation in mammals. International Journal of Molecular Sciences, 23(11): 6333. DOI:
https://www.doi.org/10.3390/ijms23116333
Douard V, Hermier D, Magistrini M, and Blesbois E (2003). Reproductive period affects lipid composition and quality of fresh and
stored spermatozoa in turkeys. Theriogenology, 59(3-4): 753-764. DOI: https://www.doi.org/10.1016/S0093-691X(02)01086-5
Dutta S, Majzoub A, and Agarwal A (2019). Oxidative stress and sperm function: A systematic review on evaluation and
management. Arab Journal of Urology, 17(2): 87-97. DOI: https://www.doi.org/10.1080/2090598X.2019.1599624
Garner D and Hafez E (2000). Spermatozoa and seminal plasma. In: B. Hafez, E. S. E. Hafez (Editors), Reproduction in farm animals,
7th Edition. Lippincont Wiliams and Wilkins, pp. 96-109. DOI: https://www.doi.org/10.1002/9781119265306.ch7
Hafez ESE and Hafez B (2000). Transport and survival of gametes. In: B. Hafez, E. S. E. Hafez (Editors), Reproduction in farm
animals, 7th Edition. Lippincont Wiliams and Wilkins, pp. 82-95. DOI: https://www.doi.org/10.1002/9781119265306.ch6
Ilaria N (2011). Sperm preparation techniques for artificial insemination - comparison of sperm washing, swim up, and density
gradient centrifugation methods. In: M. Milad (Edition), Artificial insemination in farm animals. Rijeka: IntechOpen, Chapter 7,
pp. 115-122. DOI: https://www.doi.org/10.5772/17026
Islam M, Apu A, Hoque S, Ali M, and Karmaker S (2018). Comparative study on the libido, semen quality and fertility of brahman
cross, holstein friesian cross and red chittagong breeding bulls: Libido, semen quality and fertility of breeding bulls. Bangladesh
Journal of Animal Science, 47(2): 61-67. DOI: https://www.doi.org/10.3329/bjas.v47i2.40236
Juniandri TS and Isnaini TSN (2014). Comparison of andromed and cep-2 diluter on the quality of limousin cattle spermatozoa
following sexing with percoll. Jurnal Veteriner, 15(2): 252-262. Available at:
https://ojs.unud.ac.id/index.php/jvet/article/view/9718
Lestari TP, Ihsan MN, and Isnaini N (2014). Effect of semen storage duration diluted with andromed solution at room temperature on
the quality of boer goat spermatozoa. Journal of Tropical Animal Production, 15(1): 43-50. Available at:
https://ternaktropika.ub.ac.id/index.php/tropika/article/view/196
Liu X, Xu Y, Liu F, Pan Y, Miao L, Zhu Q, and Tan S (2021). The feasibility of antioxidants avoiding oxidative damages from
reactive oxygen species in cryopreservation. Frontiers in Chemistry, 9: 648684. DOI:
https://www.doi.org/10.3389/fchem.2021.648684
Maleki K, Ayen E, Khaki A, and Soleimanzadeh A (2023). Comparison of sperm characteristics and antioxidant and oxidant levels in
bull semen frozen with four widely used extenders. Veterinary Research Forum, 14(7): 373-379. DOI:
https://www.doi.org/10.30466%2Fvrf.2023.562594.3631
Manehat FX, Dethan AA, and Tahuk PK (2021). Motility, viability, spermatozoa abnormality, and ph of bali cattle semen in another-
yellow water driller stored in a different time. Journal of Tropical Animal Science and Technology, 3(2): 76-90. DOI:
https://www.doi.org/10.32938/jtast.v3i2.1032
Mappanganro R (2020). Fresh semen production (volume and concentration) and frozen from bulls with different body condition
scores (SKT). Jurnal Ilmu dan Industri Peternakan, 6(1): 1-13. DOI: https://www.doi.org/10.24252/jiip.v6i1.14444
Murphy EM, Eivers B, O'Meara CM, Lonergan P, and Fair S (2018). Effect of increasing equilibration time of diluted bull semen up
to 72 h prior to freezing on sperm quality parameters and calving rate following artificial insemination. Theriogenology, 108:
217-222. https://www.doi.org/10.1016/j.theriogenology.2017.11.034
Nofa Y, Karja NWK, and Arifiantini RI (2017). Acrosome statusand qualityof post-thawed sperm from several cattle breed of two
artificial insemination centre. Acta Veterinaria Indonesiana, 5(2): 81-88. DOI: https://www.doi.org/10.29244/avi.5.2.81-88
Nur Z, Seven-Cakmak S, Ustuner B, Cakmak I, Erturk M, Abramson CI, Sağirkaya H, and Soylu MK (2012). The use of the hypo-
osmotic swelling test, water test, and supravital staining in the evaluation of drone sperm. Apidologie, 43: 31-38. DOI:
https://www.doi.org/10.1007/s13592-011-0073-1
Park SH and Yu IJ (2017). Effect of antioxidant supplementation in freezing extender on porcine sperm viability, motility and reactive
oxygen species. Journal of Embryo Transfer, 32(1): 9-15. DOI: https://www.doi.org/10.12750/JET.2017.32.1.9
Perumal P, Vikram R, Khate K, Vupru K, Saddamhusen M, and Khan M (2021). Glutathione in semen extender modulates post thaw
semen quality profiles, and antioxidant and oxidative stress profiles in mithun. The Indian Journal of Animal Sciences, 91(12):
1018-1026. DOI: https://www.doi.org/10.56093/ijans.v91i12.119815
Pizzorno J (2014). Glutathione. Integrative Medicine: A Clinician's Journal, 13(1): 8-12. Available at:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684116/
Prastowo S, Dharmawan P, Nugroho T, Bachtiar A, Lutojo, and Pramono A (2018). Age effect on bali cattle (bos javanicus) fresh
semen quality. Jurnal Ilmu Ternak Universitas Padjadjaran, 18(1): 1-7. DOI: https://www.doi.org/10.24198/jit.v18i1.17684
Pratiwi RI, Suharyati S, and Hartono M (2014). Analysis quality of simmental semen using andromed® extender with variations of
pre freezing time. Jurnal Ilmiah Peternakan Terpadu, 2(3): 8-15. DOI: http://www.doi.org/10.23960/jipt.v2i3.p%25p
Prihantoko KD, Kusumawati A, Pangestu M, Widayati DT, and Budiyanto A (2022). Influence of intracellular reactive oxygen
species in several spermatozoa activity in indonesian ongole bull cryopreserved sperm. American Journal of Animal and
Veterinary Sciences, 17(1): 11-18. DOI: https://www.doi.org/10.3844/ajavsp.2022.11.18
Setiono N, Suharyati S, and Santosa PE (2015). Brahman bull’s frozen semen quality with differen doses of cryprotectant glycerol in
yolk tris sitrat diluent. Jurnal Ilmiah Peternakan Terpadu, 3(2): 61-69. DOI: http://www.doi.org/10.23960/jipt.v3i2.p%25p
Solihati N, Rasad S, Setiawan R, Foziah E, and Wigiyanti E (2018). Semen quality of post-thawed local ram’s in tris-egg yolk
extender with different glutathione level. IOP Conference Series: Earth and Environmental Science, 119: 012034. DOI:
https://www.doi.org/10.1088/1755-1315/119/1/012034
Susilawati T (2011). Spermatology. Universitas Brawijaya Press, pp. 125-134.
569
To cite this paper: Bebas W, Gorda IW, Agustina KK, and Merdana IM (2023). Effects of Adding Glutathione to AndroMed Diluent on Intact Plasma and Acrosome
Membranes, and Progressive Motility of Cattle Spermatozoa During Freezing Processes. World Vet. J., 13 (4): 561-570. DOI: https://dx.doi.org/10.54203/scil.2023.wvj60
Swarna M, Biswas S, Biswas D, Saha N, and Paul A (2023). Impact of chilling duration on sperm quality of indigenous buck semen in
the coastal area of bangladesh. Bangladesh Journal of Veterinary Medicine, 21(1): 17-25. DOI:
https://www.doi.org/10.33109/bjvmjj2023fam1
Syafi'i TM and Rosadi B (2022). The ressitance of acrosome cap and plasma membrane of bali cattle spermotozoas exposed to room
temperature. Jurnal Produksi Ternak Terapan, 3(2): 41-46. DOI: https://www.doi.org/10.24198/jptt.v3i2.42471
Syafitri M, Prabowo TA, Sitaresmi PI, Yusiati LM, Bintara S, and Widayati DT (2022). The effect of glutathione addition in diluent
semen on ram spermatozoa quality. 9th International Seminar on Tropical Animal Production. Atlantis Press, pp. 251-255. DOI:
https://www.doi.org/10.2991/absr.k.220207.052
Syarifuddin A, Laksmi DNDI, and Bebas IW (2012). Eeffectiveness of adding various glutathione concentrations on viability and
motility of post thawing bali cattle spermatozoa. Indonesia Medicus Veterinus, 1(2): 173-185. Available at:
https://ojs.unud.ac.id/index.php/imv/article/view/1806
Wishart GJ (2009). Semen quality and semen storage. Poultry Science Symposium Series, Edinburgh, UK, pp. 151-178. DOI:
https://www.doi.org/10.1079/9781845933753.0151
Zeitoun MM and Al-Damegh MA (2014). Effect of nonenzymatic antioxidants on sperm motility and survival relative to free radicals
and antioxidant enzymes of chilled-stored ram semen. Open Journal of Animal Sciences, 5(1): 50-57. DOI:
https://www.doi.org/10.4236/ojas.2015.51007
Zhang B, Wang Y, Wu C, Qiu S, Chen X, Cai B, and Xie H (2021). Freeze-thawing impairs the motility, plasma membrane integrity
and mitochondria function of boar spermatozoa through generating excessive ros. BMC Veterinary Research, 17(1): 127. DOI:
https://www.doi.org/10.1186/s12917-021-02804-1
Zhang H, Forman HJ, and Choi J (2005). Γ‐glutamyl transpeptidase in glutathione biosynthesis. Methods in Enzymology, 401: 468-
483. DOI: https://www.doi.org/10.1016/S0076-6879(05)01028-1
Zou J, Wei L, Li D, Zhang Y, Wang G, Zhang L, Cao P, Yang S, and Li G (2021). Effect of glutathione on sperm quality in
guanzhong dairy goat sperm during cryopreservation. Frontiers in Veterinary Science, 8: 771440. DOI:
https://www.doi.org/10.3389/fvets.2021.771440
Zulaikhah ST (2017). The role of antioxidant to prevent free radicals in the body. Sains Medika Journal of Medical & Health, 8(1):
39-45. DOI: https://www.doi.org/10.26532/sainsmed.v8i1.1012
Publisher’s note: Scienceline Publication Ltd. remains neutral with regard to jurisdictional claims in published maps and institutional
affiliations.
Open Access: This article is licensed under a Creative Commons Attribution 4.0 International License, which permits
use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit
to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The
images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in
a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
To view a copy of this licence, visit https://creativecommons.org/licenses/by/4.0/.
570
To cite this paper: Bebas W, Gorda IW, Agustina KK, and Merdana IM (2023). Effects of Adding Glutathione to AndroMed Diluent on Intact Plasma and Acrosome
Membranes, and Progressive Motility of Cattle Spermatozoa During Freezing Processes. World Vet. J., 13 (4): 561-570. DOI: https://dx.doi.org/10.54203/scil.2023.wvj60